Reduction of the electrocardiographic ot interval

ABSTRACT

The present invention relates to the use of proinsulin C-peptide or a variant, derivative or fragment thereof in the manufacture of a medicament for reducing the OTc interval and the use of proinsulin C-peptide or a variant, derivative or fragment thereof in the manufacture of a medicament for reducing the risk of sudden death or ‘dead in bed’ syndrome; particularly in patients suffering from IDDM.

[0001] The present invention relates to therapies whereby QT intervalprolongation is normalised and the risk of sudden death or ‘dead in bed’syndrome is reduced.

[0002] It has been observed (Davidson's Principles and Practice ofMedicine, 17th Ed., p. 266) that a family history of sudden death inchildhood or young adult life is sometimes associated with prolongationof the QT interval on the ECG (electrocardiogram). More specifically,several recent studies have shown that patients with insulin dependentdiabetes mellitus (IDDM) have a higher prevalence of QT intervalprolongation (Lengyel et al. Orv Hetil 1997; 138(6): 337-41). The QTinterval reflects the time between the start of electrical activation ofthe myocardial ventricular wall and completion of its repolarization. QTduration is dependent on the heart rate, when corrected for heart rateinfluence, QT is denoted QTc. QTc is prolonged among patients withischaemic heart disease (Ahnve et al., American Heart Journal 1984; 108:395-400), diabetes mellitus (Sawicki et al., Diabetologia 1996; 39:77-81) and after modulation of the autonomic nervous system with drugs(Tentolouris et al., Eur. J. Clin. Invest. 1997; 27(12): 1049-54). Thepathogenesis of QTc prolongation is not known and no medication for ortreatment of prolonged QTc is currently available.

[0003] QTc prolongation, known to be associated with arrhythmias, mayhave a role in the ‘dead in bed’ syndrome of insulin-dependent diabetesmellitus (IDDM) patients, in which seemingly healthy diabetic patientsdie unexpectedly without immediate cause. A preliminary study(Tattersall et al., Diabet. Med. 1991; 8: 49-58) reported a series of 22unexplained deaths in patients with IDDM, all of whom were less than 50years old. The deaths followed a well defined pattern, they occurred atnight and the patients were found the next morning in an undisturbedbed. They had been seen the day before in good health and the autopsyconsistently revealed no anatomical cause for death. Only a smallminority of patients had documented ante-mortem evidence of diabeticcomplications, and in only one patient was there definite evidence ofautonomic neuropathy. The phenomen became known as the ‘dead in bedsyndrome’. Since 1991 more than 80 young diabetic patients have died insudden death syndrome in Scandinavia alone (Weston et al., Diabet. Med.1999; 16(8): 626-31). It is therefore important to establish the causeand even more important to find a cure. QTc prolongation might bedecreased with angiotensin-converting enzyme inhibitors or β-blockers,but QTc prolongation is not a recognised indication for administrationof these drugs which have significant adverse effects.

[0004] The present invention is based on the surprising observation thatproinsulin C-peptide is able to reduce the QTc interval in patientsexhibiting QTc interval prolongation. Given the documented correlationbetween QTc interval prolongation and ‘dead in bed’ syndrome, thisobservation suggests a novel prophylactic treatment of ‘dead in bed’syndrome. In particular the QTc interval can be reduced in patients withIDDM.

[0005] C-peptide has long been thought not to have any biologicalactivity. It is synthesised in the beta cells of the pancreas andreleased into the circulation in equimolar amounts together withinsulin. The C-peptide amino acid chain is a part of the proinsulinmolecule and it is thought to assist in achieving the correct foldingstructure. When the proteolytic process starts the proinsulin is cleavedinto insulin and C-peptide. Human C-peptide is a 31 amino acid peptidehaving the following sequence:

[0006] C-peptide binds to the cell surface and stimulates Na⁺K⁺-ATPaseand endothelial nitric oxide synthase (eNOS) activity, probably via aG-protein coupled reaction followed by activation of Ca²⁺-dependentintracellular signaling pathways. Studies have shown that administrationof C-peptide can decrease glomerular hyperfiltration, improveblood-retinal barrier function (Johansson et al., J. Clin. Endocrinol. &Metab. 1993; 77: 976-981) and ameliorate autonomic and sensory nervefunction in patients with IDDM (Johansson et al., Diabetologia 1996; 39:687-695). Active fragments of C-peptide have been described for treatingdiabetic neuropathy, nephropathy and retinopathy (W098/13384 but not forimproving myocardial complications of diabetes such as an elongated QTcinterval. A proportion, perhaps 2-10%, of patients with IDDM willexhibit an elongated QTc interval but this group will not necessarilyalso exhibit diabetic neuropathy/nephropathy etc., there being no knownassociation between the conditions.

[0007] Thus, in one aspect the present invention provides the use ofproinsulin C-peptide in the manufacture of a medicament for reducing theQTc interval, typically in patients exhibiting QTc intervalprolongation. Generally the patients will have IDDM. However, QTcinterval prolongation is also observed in other patients such as thosewith ischaemic heart disease and those who have received drugs whichmodulate the autonomic nervous system and such patients may also benefitfrom the treatments discussed herein.

[0008] Alternatively viewed, the invention provides proinsulin C-peptidefor use in reducing the QTc interval, typically in patients exhibitingQTc interval prolongation, generally in patients with IDDM.

[0009] In a further aspect, the present invention provides the use ofproinsulin C-peptide in the manufacture of a medicament for theprophylactic treatment (i.e. prevention, including reducing the risk) ofsudden death or ‘dead in bed’ syndrome. This syndrome is discussed aboveand patients who are believed to be at risk of ‘dead in bed’ syndromewill typically be those exhibiting QTc interval prolongation, generallypatients with IDDM.

[0010] Alternatively viewed the invention provides proinsulin C-peptidefor use in the prophylactic treatment of sudden death or ‘dead in bed’syndrome.

[0011] According to a further aspect, the present invention provides amethod of reducing the QTc interval in a mammal, which method comprisesadministering proinsulin C-peptide to said mammal in an amount effectiveto reduce said QTc interval.

[0012] In a yet further aspect, the present invention provides a methodof preventing or reducing the risk of sudden death or ‘dead in bed’syndrome in a human or animal patient who is at risk thereof, whichmethod comprises administering proinsulin C-peptide in an amount effectto reduce the QTc interval in said patient. Patients at risk of ‘dead inbed’ syndrome can be identified as those exhibiting QTc intervalprolongation and will typically be sufferers of IDDM.

[0013] The invention relates to human proinsulin C-peptide itself aswell as functionally equivalent variants, derivatives or fragmentsthereof. Such variants may include, for example, different allelicvariants as they appear in nature, e.g. in other species or due togeographical variation etc. Functionally equivalent derivatives includepeptides which incorporate amino acid substitutions, or intra-sequenceor terminal deletions or additions to the above sequence, and alsochemical modifications thereof, including for example the inclusion ofchemically substituted or modified amino acid residues, provided theyretain the ability to reduce the QTc interval. The variants, derivativesand fragments are functionally equivalent in that they exhibit at least40%, preferably at least 60% of the activity of human proinsulinC-peptide. Activity being measured in terms of an ability to reduce theQTc interval as described herein.

[0014] Also included within the scope of the invention is the use of“non-native” isomers of “native” L-amino acid C-peptide sequences, e.g.peptides containing D-amino acid isomers.

[0015] It is known in the art to modify the sequences of proteins orpeptides, whilst retaining their useful activity, and this may beachieved using techniques which are standard in the art and widelydescribed in the literature e.g. random or site-directed mutagenesis,cleavage and ligation of nucleic acids, chemical peptide synthesis etc.

[0016] A peptide may conveniently be chosen which is human proinsulinC-peptide or a functional fragment thereof or a functional peptide whichhas an amino acid sequence which is 70%, preferably 80%, more preferably90% identical to C-peptide or a fragment thereof.

[0017] As far as fragments are concerned, these include both fragmentsof the native human proinsulin C-peptide sequence as set out above, orfragments of any functionally equivalent variant or derivative asmentioned above, provided that the fragment retains the biological ortherapeutically beneficial activity of the “whole” or “complete”molecule. Preferred fragments comprise residues 15-31 of nativeC-peptide, more especially residues 20-31. Peptides comprising thepentapeptide EGSLQ (residues 27-31 of native human C-peptide) areparticularly preferred. The fragment may thus vary in size from e.g. 2to 30 amino acids, preferably 3 to 20, e.g. 3 to 12, or 4 to 10residues. The fragments will typically have at least 5 amino acidresidues, e.g. 5 to 12 residues.

[0018] Representative fragments include or comprise in particularpeptide A (ELGGGPGAG) or peptide E (EGSLQ) mentioned in W098/13384 ofCreative Peptides Sweden AB, sub-fragments of peptides A and E may alsobe used and these are described in more detail in WO 98/13384. Exemplarymention may be made of peptide B (ELGG), peptide C (ELGGGP), peptide D(GGPGA) and peptide F (GSLQ). Also included with the scope of definitionof “proinsulin-C peptide” according to the present invention arepeptides having N- and/or C-terminal extensions; or flanking sequences,to the sequences of proinsulin C-peptide or a fragment, thereof. Suchflanking or extension sequences may be non-native. The length of each“extended” derivative may vary, but preferably the peptides are not morethan 50, e.g. not more than 30, or 25 or 20, especially not more than 15or 10 amino acids in length. For example, the peptides may be 3 to 50, 3to 30, 3 to 25, 3 to 20, 3 to 15 or 3 to 10 amino acids in length.

[0019] Accordingly, by “proinsulin C-peptide” is meant native, isolatedproinsulin C-peptide whether modified or unmodified, as well assynthesised peptides, and all such variants, derivatives and fragmentsas are described above.

[0020] In addition, the term “proinsulin C-peptide” encompassesnon-peptide compounds showing the same effects as displayed by theirC-peptide-derived counterparts. Such peptidomimetics or“small-molecules” capable of mimicking the activity of the naturallyoccurring proteins or peptides are likely to be better suited for e.g.oral delivery due to their increased chemical stability.

[0021] It is now commonplace in the art to replace peptide orprotein-based active agents e.g. therapeutic peptides, with suchpeptidomimetics having functionally-equivalent activity. Variousmolecular libraries and combinatorial chemistry techiques exist and areavailable to facilitate the identification, selection and/or synthesisof such compounds using standard techniques (Kieber-Emons, T. et al.,Current Opinion in Biotech. 1997; 8: 435-441). Such standard techniquesmay be used to obtain the peptidomimetic compounds for use according tothe present invention, namely peptidomimetic organic compounds whichshow the same or substantially similar or even better reduction of theQTc interval, e.g. as is shown by the peptides described herein in theExamples.

[0022] The suitability of a given proinsulin C-peptide for use accordingto the invention can be determined by its ability to reduce the QTcinterval according to the protocol described in the Examples herein.

[0023] The QTc interval will vary from patient to patient but cangenerally be considered prolonged when it exceeds 420 ms in men and 430ms in women. QTc is the corrected (for heart rate influence) QTinterval. It is calculated according to Bazett's formula (QTc=QT/{squareroot}RR). QT and RR are conveniently measured using a ruler on an ECGtracing; the QT interval is measured from the beginning of the QRScomplex to the end of the crossing of the isoelectric line of the Twave, see the stylised representation in FIG. 1. The RR interval ismeasured from one R peak to the next, again as shown in FIG. 1. Anyreduction in the interval can be beneficial to the patient, preferablythe interval will be reduced by at least 3 ms, more preferably treatmentwill reduce the QTc interval by at least 5 ms, particularly preferablyat least 8 ms even at least 10 ms.

[0024] In addition, many drugs administered to diabetic patientsincrease the QTc interval so even a modest decrease may be clinicallyimportant.

[0025] Patients with IDDM generally have very low circulating levels ofproinsulin C-peptide and the therapies of the present inventionpreferably result in a normalisation of circulating proinsulin C-peptidelevels. Preferably the patient will have 60 to 140, e.g. 80-120% of thenormal physiological level of circulating proinsulin C-peptide as aresult of the administration in accordance with the present invention.

[0026] Compositions comprising proinsulin C-peptide as defined above arepreferably formulated prior to administration.

[0027] The present invention therefore also provides a pharmaceuticalcomposition for use in the reduction of the QTc interval and/or theprevention of ‘dead in bed’ syndrome (sudden death), said compositioncomprising proinsulin C-peptide together with at least onepharmaceutically acceptable carrier, diluent or excipient.

[0028] The active ingredient in such compositions may comprise from0.05% to 99% by weight of the formulation, more preferably 0.1% to 1.0%.

[0029] By “pharmaceutically acceptable” is meant that the ingredientsmust be compatible with other ingredients of the composition as well asphysiologically acceptable to the recipient.

[0030] Pharmaceutical compositions for use in methods according to thepresent invention may be formulated according to techniques andprocedures well known in the art and widely described in the literature,and may comprise any of the known carriers, diluents or excipients.Other ingredient may of course also be included, according to techniqueswell known in the art e.g. stabilisers, preservatives, etc. Theformulations may be in the form of sterile aqueous solutions and orsuspensions of the pharmaceutically active ingredients, aerosols,ointments and the like.

[0031] A preferred embodiment of the present invention are sustainedrelease forms of proinsulin C-peptide which are well known in the art,e.g. microparticles, nanoparticles, emulsions, nanosuspensions, lipidparticles or oils. Especially preferred are sustained release formsbased on microparticles.

[0032] Materials used for the production of said microparticles are wellknown in the art and comprise e.g. mono- or copolymers based on lactite,glycolite, vinyl acetate, dextrans, dextransulfates, hydroxybutyrate,valerolacton, caprolacton, acrylic acid or methacrylic acids. Especiallypreferred are microparticles based on polylactite or polyglycolite.Examples for the production of such particles are given in patentapplication No. WO 94/09898.

[0033] Another preferred embodiment of the present invention are films,patches or folios having proinsulin C-peptide coated on the surface,incorporated in a special layer or incorporated in the matrixcomposition. An especially preferred embodiment are biodegradable folioshaving proinsulin C-peptide coated on the surface or incorporated in thecomposition. Preferred materials of said preferred biodegradable foliosare polymers or copolymers of lactite and glycolite.

[0034] The administration may be by any suitable method known in themedicinal arts, including oral, parenteral, topical, subcutaneousadministration or by inhalation.

[0035] The peptides may be administered in a single dose to be taken atregular intervals, or as divided doses to be taken/administered e.g. 1to 6 times during the course of a day. Sustained release formulationsare preferably given at longer intervals, e.g. 1 to 2 times a month orevery three months.

[0036] The precise dosage of the active compounds to be administered,the number of daily or monthly doses and the length of the course oftreatment will depend on a number of factors, including the age of thepatient and the degree of QTc interval prolongation.

[0037] Conveniently, the C-peptide may be administered intravenously orsub-cutaneously. Typical doses will range from 300-1000 nM per day, e.g.500-800 rM per day, this total dose preferably being split between 2 ormore doses during the 24 hour period, typically being split between 3 or4 doses. Patients who would benefit from the treatments proposed hereinwill often be those who take several daily doses of insulin (e.g. bysub-cutaneous self injection) and administration of C-peptide can followa similar pattern. Insulin and C-peptide may therefore effectively beadministered at the same or substantially the same time, but preferablynot in a mixed formulation. As the C-peptide may be administered byinjection, the ‘medicament’ comprising it will typically be in liquidform comprising one or more solvents as well as the activeingredient(s). The treatment program will follow the typical pattern ofchronic treatment and is likely to last for months, possibly for thelife of the patient.

[0038] The compositions may be formulated according to techniques andprocedures well known in the literature, and may comprise any of theknown carriers, diluents or excipients. Thus, for example, compositionsfor use in the methods of this invention which are suitable forparenteral administration conveniently comprise sterile aqueoussolutions and/or suspensions of pharmaceutically active ingredientspreferably made isotonic with the blood of the recipient, generallyusing sodium chloride, glycerin, glucose, mannitol, sorbitol and thelike. In addition the composition may contain any of a number ofadjuvants, such as buffers, preservatives, dispersing agents, agentsthat promote rapid onset of action or polonged duration of action andthe like.

[0039] Compositions suitable for oral administration may, for example,comprise active fragments/peptides of the proinsulin C-peptide moleculeor the whole molecule in sterile purified stock powder form, preferablycovered by an envelope or envelopes (enterocapsulae) protecting theactive peptides from degradation (decarboxylation or hydrolysis) in thestomach and thereby enabling absorption of these substances from thegingiva or in the small intestine. The envelope(s) may contain any of anumber of adjuvants such as buffers, preservative agents, agents thatpromote prolonged or rapid release giving an optimal bioavailability ofthe compositions. Furthermore, compositions for use in the methods ofthis invention suitable for local or topical administration may compriseactive fragments of the proinsulin C-peptide molecule or the wholemolecule in sterile formulation mixed with known suitable ingredients,such as paraffin, vaseline, cetanol, glycerol and its like, to formsuitable ointments or creams.

[0040] The invention will now be described in more detail in thefollowing non-limiting Examples which show, with reference to thefollowing drawings:

[0041]FIG. 1 is a schematic representation of a typical ECG traceshowing the QT and RR intervals. The P wave represents atrialactivation, the QRS complex ventricular activation and the T waveventricular recovery.

[0042]FIG. 2 is a graph showing the influence of proinsulin C-peptide onQTc intervals in seconds according to the study described in Example 1herein.

[0043]FIG. 3 is a graph showing the influence of proinsulin C-peptide onQTc interval in 6 patients with IDDM, after 3 months of treatment asdescribed in Example 2.

[0044]FIG. 4 is a graph showing the influence of proinsulin C-peptide onQTc interval in 13 patients with IDDM, after 3 hours infusion asdescribed in Example 3. The 13 patients are made up of the 8 patients ofExample 1 plus the 5 (from 12) patients of Example 3 who exhibit QTcprolongation.

[0045]FIG. 5 is a graph showing the differences in QTc interval betweenthe C-peptide and placebo (NaCl) infusion periods in percentage (%) andin real terms (ms) for the 13 patients of FIG. 4 described above plusthe 6 patients of Example 2.

[0046]FIG. 6 is a graph showing the influence of proinsulin C-peptideand placebo on the QTc interval of the patients with prolonged basalQTc. This group of 19 patients is the same as described above in respectof FIG. 5.

EXAMPLE 1

[0047] Electrocardiograms from a study by Johansson et al. carried outin 1996 (other aspects of this study were reported in Johansson et al.,1996, supra) were examined and this paper is incorporated herein byreference.

[0048] In this study 12 IDDM patients (six male and six female) withautonomic neuropathy participated in two different sessions in arandomized, double-blind study (one patient attended one time only).Their mean age was 40 and mean duration of diabetes was 21 years. Ineight patients the fasting plasma C-peptide levels were below thedetection limit (<0.10 nmol/l), the remaining four varied between 0.13and 0.25 nmol/l. Eleven patients had decreased heart rate variabilityduring deep breathing as evidence of autonomic neuropathy. On eachoccasion they received insulin i.v. for 9-12 hours before the study andblood glucose levels were tested every 1-2 hours during the night. Theinsulin infusion was adjusted to achieve euglycaemia (5-6 mmol/l). Thepatients were then given either saline or C-peptide i.v. for 180minutes. 6 pico mole per kg per minute was administered.

[0049] Before and during the three hour session a complete 12 leadelectrocardiogram was recorded to evaluate heart rate variability duringdeep breathing. These recordings were then used to measure the correctedQT interval (QTc) according to the Bazett's formula (QTc=QT/{squareroot}RR). The 44 ECG tracings (one patient could not take part in asecond session and one patient's ECG tracing was not possible toevaluate) were analyzed blindly by two persons. The RR and QT intervalwere measured with a ruler. The QT interval was taken from the beginningof the QRS complex to the crossing of the isoelectric line of the Twave. Five intervals from the precordial leads and five intervals fromthe extremity leads were measured on each occasion and a mean wascalculated. The two observers compared their QTc values if there was adifference greater than 5%, it was recalculated. P values less than 0.05were considered statistically significant.

[0050] Mean C-peptide levels rose from 0.11±0.02 nmol/l before the studyto 1.73±0.04 nmol/l during the C-peptide infusion (within normalphysiological range), whereas the C-peptide plasma concentrationremained below detection limit (0.10 nmol/l) during saline infusion.Blood glucose levels decreased slightly during C-peptide infusion (from6.3 to 5.9 mmol/l) and during the saline infusion it was almostunchanged. There was no difference in the basal QTc interval on the twostudy occasions (0.429±0.004s). During the three hours of C-peptideadministration the mean QTc interval decreased significantly(0.422±0.006; p<0.05), whereas the QTc interval rose slightly during thesaline infusion (n.s.) (FIG. 2). This study demonstrates that infusionof C-peptide significantly decreases the QTc interval. Although thereduction is small in percentage terms the drop is significant inphysiological and clinical terms.

EXAMPLE 2

[0051] In a further study (different results from which were publishedin Diabetic Medicine, Vol. 17 (2000) p. 181-189, Johannson, Bo-Lennartet al.) 21 IDDM patients were monitored for 6 months according to arandomised cross over regimen wherein C-peptide was administered for 3months and a placebo.

[0052] Further details of the study can be found in the above referencedpaper in Diabetic Medicine but in summary, biosynthetic human C-peptide(Eli Lilly Co.) or a placebo comprising just the C-peptide solvent wasinjected sub-cutaneously 3 times per day. The total 24 h dose ofC-peptide was 600 nmol (225 nmol 30 mins before breakfast, 150 nmolbefore dinner and 225 nmol at bedtime).

[0053] ECGs were recorded at the start of the programme and after 3 andthen 6 months of the study. These recordings were then used to measurethe QTc interval as described in Example 1.

[0054] Those patients (6) who exhibited a prolonged QTc interval at theoutset (>420 ms) showed a clear shortening of the QTc interval duringthe C-peptide phase as compared to the placebo phase (FIG. 3).

EXAMPLE 3

[0055] In a further study, it has been shown that a short period ofC-peptide administration may still be sufficient to reduce the QTcinterval.

[0056] Twelve patients were investigated twice in a randomised doubleblind study where C-peptide or a NaCl placebo were infused for 3 hours.The same procedure as described in Example 1 was followed.

[0057] The ECGs were analysed as described in the previous examples anda decrease in QTc interval during C-peptide treatment as compared toduring administration of a placebo was observed as shown in FIG. 4.

[0058] The differences in QTc interval between the C-peptide and NaClinfusion periods in percentage and in real time (ms) are shown in FIG.5.

1. Use of proinsulin C-peptide or a variant, derivative or fragmentthereof in the manufacture of a medicament for reducing the QTcinterval.
 2. Use of proinsulin C-peptide or a variant, derivative orfragment thereof in the manufacture of a medicament for reducing therisk of sudden death or ‘dead in bed’ syndrome.
 3. A use as claimed inclaim 1 or claim 2 wherein the medicament is administered to a patientexhibiting QTc interval prolongation.
 4. A use as claimed in anypreceding claim wherein the medicament is administered to a patient withinsulin-dependent diabetes mellitus or ischaemic heart disease.
 5. A useas claimed in any preceding claim wherein the QTc interval is reduced byat least 5 ms.
 6. A use as claimed in any preceding claim wherein themedicament contains 100-300 nM of proinsulin C-peptide or a variantderivative or fragment thereof.
 7. A use as claimed in any precedingclaim wherein the medicament is capable of providing a daily dose of500-700 nM of pro-insulin C-peptide or a variant, derivative or fragmentthereof.
 8. A method of reducing the QTc interval in a mammal, whichmethod comprises administering proinsulin C-peptide or a variant,derivative or fragment thereof to said mammal in an amount effective toreduce said QTc interval.
 9. A method of preventing or reducing the riskof sudden death or ‘dead in bed’ syndrome in a human or animal patientwho is at risk thereof, which method comprises administering proinsulinC-peptide or a variant, derivative or fragment thereof in an amounteffect to reduce the QTc interval in said patient.